The invention relates generally to methods for detecting indicia of cancer in biological samples. In particular, the invention relates to methods for detecting the presence of hypermethylated nucleic acids in heterogeneous biological samples.
In higher order eukaryotes, DNA may be methylated at cytosines located 5xe2x80x2 to guanosine in CpG dinucleotides. This modification has important regulatory effects on gene expression, especially when involving CpG rich areas, known as CpG islands, often found in the promoter regions of many genes. Aberrant methylation of normally unmethylated CpG islands has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers, e.g., colorectal cancer. Therefore, detection of hypermethylated nucleic acid can indicate the onset of various forms of cancers.
Various methods have been described for detecting hypermethylation at specific loci on genomic DNA. One of these methods involves modification of DNA by sodium bisulfite or a comparable agent which converts all unmethylated cytosines to uracils without converting methylated cytosines, and subsequent amplification with primers specific for methylated versus unmethylated DNA. However, this and other hypermethylation detection methods require homogeneous nucleic acid samples that have a sufficient amount of the target nucleic acid. For many types of cancers, invasive procedures such as surgery are needed to obtain such a homogenous sample.
However, many biological samples, such as stool samples, are heterogeneous and may contain a small amount of mutant nucleic acid mixed with a large amount of wild-type nucleic acid. Therefore, there is a need in the art for methods to detect hypermethylation in heterogeneous biological samples.
Methods of the invention make it possible to detect a disease in biological samples without using an invasive procedure. According to the invention, by conducting a hypermethylation detection assay on a heterogeneous nucleic acid sample, such as a sample obtained from stool, small amounts of hypermethylated nucleic acids can be detected that are indicative of the presence of a disease, e.g., cancer or precancer, in a patient. In one aspect, methods of the invention involve detecting small amounts of hypermethylated nucleic acid by improving the efficiency and specificity of nucleic acid amplification. In another aspect, methods of the invention also use a combination of target CpG markers or loci to improve the reliability of detecting disease-associated hypermethylation.
In one aspect, methods of the invention comprise the utilization of improved primers for detecting hypermethylation in a target nucleic acid. In a preferred embodiment, methods of the invention comprise conducting a nucleic acid amplification using one or more (preferably two) chimeric primers. A chimeric primer, for purposes of the invention, is a primer having a 3xe2x80x2 portion with substantial sequence specificity with the target template to be amplified (a template-specific portion) and a 5xe2x80x2 portion that is referred to herein as a xe2x80x9cnon-specific portionxe2x80x9d that does not hybridize with the target template. The 5xe2x80x2 non-specific sequence preferably includes at least 5, but fewer than or equal to 30 nucleotides. Also, the 5xe2x80x2 non-specific portion preferably has a G-C content of at least 50%. In a particularly preferred embodiment, the 5xe2x80x2 non-specific portion of both the forward and the reverse primers are the same. The target nucleic acid may be DNA, RNA or a nucleic acid analog. Also in a preferred embodiment, the target nucleic acid is isolated from a stool sample.
In another aspect, methods of the invention comprise the optimization of reaction conditions for PCR to detect hypermethylation in a subpopulation of nucleic acids in a biological sample. In a preferred embodiment, methods of the invention comprise determining a first melting temperature for a primer in a PCR that amplifies a target nucleic acid, and conducting a PCR at a second melting temperature that is higher than the first melting temperature, using at least one chimeric primer. The melting temperature, sometimes called the annealing temperature, as referred to herein, means the temperature at which the strands of a nucleic acid hybrid are about 50% dissociated. The second melting temperature is preferably more than about 3xc2x0 C. higher than the first melting temperature. In a highly preferred embodiment, the second melting temperature is about 65xc2x0 C.
In another aspect, the invention provides methods for screening a population to detect patients with indicia of cancer. In preferred embodiments, a plurality of target CpG containing regions are interrogated for the presence of hypermethylation. Preferably, one or more of the following regions are assayed in patient nucleic acid isolated from a heterogeneous biological sample such as stool: HIC1, p14, HLTF, MINT2, and MINT31 regulatory regions. The presence of hypermethylation in at least one of these regions is indicative of the presence of indicia of cancer in the patient. According to the invention, hypermethylation is an amount of methylation that is greater than that found in normal tissue or normal cells isolated from a healthy patient. Accordingly, preferred methods of the invention include a negative control assay using nucleic acid from normal cells. In more preferred embodiments, methods of the invention also include a positive control assay using hypermethylated nucleic acid.
In a further aspect, methods of the invention are also useful to detect hypermethylation in a substantially homogeneous sample such as a biopsy sample.
These and other advantages and aspects of the invention will be understood upon consideration of the following detailed description thereof.